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Genetic analysis of Phytophthora infestans populations in theNordic European countries reveals high genetic variability

May Bente BRURBERGa,*, Abdelhameed ELAMEENa, Vinh Hong LEa, Ragnhild NÆRSTADa,Arne HERMANSENa, Ari LEHTINENb, Asko HANNUKKALAb, Bent NIELSENc,Jens HANSENd, Bj€orn ANDERSSONe, Jonathan YUENe

aNorwegian Institute for Agricultural and Environmental Research, Plant Health and Plant Protection Division, Bioforsk,

Høgskoleveien 7, 1432 �As, NorwaybMTT Agrifood Research Finland, Plant Production Research, FI-31600 Jokioinen, FinlandcAarhus University, Department of Integrated Pest Management, Research Centre Flakkebjerg, Forsoegsvej 1, DK-4200 Slagelse, DenmarkdAarhus University, Department of Agroecology and Environment, Research Centre Foulum, P.O. Box 50, 8830 Tjele, DenmarkeSwedish University of Agricultural Sciences, Department of Forest Mycology and Pathology, P.O. Box 7026, S-750 07 Uppsala, Sweden

a r t i c l e i n f o

Article history:

Received 6 January 2010

Received in revised form

4 January 2011

Accepted 17 January 2011

Corresponding Editor:

Hermann Voglmayr

Keywords:

Oomycete

Phytophthora infestans

Population genetics

Simple-sequence repeat (SSR)

* Corresponding author. Tel.: þ47 92609364; fE-mail address: may.brurberg@bioforsk.n

1878-6146/$ e see front matter ª 2011 The Bdoi:10.1016/j.funbio.2011.01.003

Please cite this article in press as: Brurbepean countries reveals high genetic varia

a b s t r a c t

Late blight, caused by the oomycete Phytophthora infestans, is the most important disease of

potato (Solanum tuberosum). The pathogen is highly adaptable and to get an overview of the

genetic variation in the Nordic countries, Denmark, Finland, Norway and Sweden we have

analyzed 200 isolates from different fields using nine simple-sequence repeat (SSR)

markers. Forty-nine alleles were detected among the nine SSR loci and isolates from all

four Nordic countries shared the most common alleles across the loci. In total 169 multi-

locus genotypes (based on seven loci) were identified among 191 isolates. The genotypic di-

versities, quantified by a normalized Shannon’s diversity index (Hs), were 0.95 for the four

Nordic countries. The low FST value of 0.04 indicates that the majority of variation is found

within the four Nordic countries. The large number of genotypes and the frequency distri-

bution of mating types (60 % A1) support the hypothesis that sexual reproduction is con-

tributing notably to the genetic variation of P. infestans in the Nordic countries.

ª 2011 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introduction P. infestans is hemibiotrophic and the pathogen generally

The oomycete Phytophthora infestans that causes late blight in

potato (Solanum tuberosum) and tomato (Lycopersicon esculen-

tum) is considered one of the world’s most devastating plant

pathogens. Late blight in potatoes led to the Irish potato fam-

ine in the mid-1840s, which resulted in the death and dis-

placement of millions of people, and this disease is still

among the worst crop diseases of the world despite much re-

search efforts over the years (recently reviewed by Fry 2008).

ax: þ47 64946110.oritish Mycological Societ

rg MB, et al., Genetic anability, Fungal Biology (2

survives between crop seasons in potato tubers. P. infestans

spreads asexually via sporangia, which are dispersed by water

or wind, hence having the potential to spread over longer dis-

tances (Aylor 2003). P. infestans is diploid and heterothallic

with two knownmating types, A1 and A2. Interaction between

hyphae of opposite mating type induces the formation of an-

theridia and oogonia that may associate and fuse to form an

oospore, which means that the pathogen has the potential

to reproduce sexually. In contrast to sporangia, oospores are

y. Published by Elsevier Ltd. All rights reserved.

lysis of Phytophthora infestans populations in the Nordic Euro-011), doi:10.1016/j.funbio.2011.01.003

2 M. B. Brurberg et al.

tolerant of adverse conditions and can survive in soil between

growing seasons (Drenth et al. 1995).

Until the mid-1970s, isolates of P. infestans found outside

North America were generally considered to belong to the

US-1 clonal lineage (mating type A1; Goodwin et al. 1994), but

during the 1980s, a novel mating type, A2, was detected in

Europe (Hohl & Iselin 1984). The appearance of mating type

A2 could, in principle, allow P. infestans to reproduce sexually,

with subsequent effects on disease epidemiology and control.

Firstly, oospores represent a stable resting phase that is inde-

pendent of the host. Secondly, sexual reproduction is likely to

increase the adaptability of the organism. Interestingly, in the

last two decades, newmore virulent strains and increased fre-

quencies of fungicide resistance have been observed (Fry

2008). There are now several studies showing that the

pathogen population in Europe is becoming increasingly

diverse (Drenth et al. 1994; Sujkowski et al. 1994; Andersson

et al. 1998; Brurberg et al. 1999; Hermansen et al. 2000;

Turkensteen et al. 2000; Flier et al. 2007; Widmark et al. 2007).

Over the years, a range of markers, both phenotypic and

genotypic, have been used for studying genetic variation in

P. infestans (reviewed by Cooke& Lees 2004). Standardized, val-

idated simple-sequence repeat (SSR) protocols for improved

comparison between P. infestans isolates, across labs

and countries, as well as a database with genotypic data

have made SSR-based techniques the tool of choice for

studying genetic variation in P. infestans (Lees et al. 2006;

www.eucablight.org).

Since P. infestans remains a major pathogen showing in-

creasing adaptability, it is interesting to monitor its diversity,

especially in areaswhere this diversity seems to be high. Previ-

ous studies on Nordic potato crops have revealed considerable

diversity (Andersson et al.1998; Brurberg et al.1999;Hermansen

et al.2000; Flier et al.2007;Widmark et al.2007) leading to thehy-

pothesis that sexual reproduction has been going on for more

than a decade already, at least in the regions that were ana-

lyzed in previous studies. To get better insight into Nordic di-

versity and to analyze the consequences of more than one

decade of (presumed) sexual reproduction, we have now con-

ducteda largestudyongeneticdiversity ofP. infestans, covering

all important potato growing regions in the Nordic countries.

We have used the highly reproducible and high-throughput

SSR-based technique for analyzing genetic variation in a large

collection of recent isolates spanning over four Northern Euro-

pean countries, Denmark, Finland, Norway, and Sweden.

Fig 1 e Location of the potato crops from which the 200

Phytophthora infestans isolates were sampled.

Materials and methods

Collection and culturing of isolates of Phytophthorainfestans

During summer 2003, 743 isolates of P. infestanswere collected

from 320 potato fields, both conventional and organic, in the

four Nordic countries (Lehtinen et al. 2008). Isolates were

obtained from leaveswith single lesions, andweremainly col-

lected when approximately 10 % of the leaf area was affected

by blight. Details regarding isolations and phenotypic charac-

terizations, including determination of mating types, have

been described by Lehtinen et al. (2008). Axenic isolates were

Please cite this article in press as: Brurberg MB, et al., Genetic anapean countries reveals high genetic variability, Fungal Biology (2

kept on different agar media (Lehtinen et al. 2008) at 18 �C in

the dark and were transferred every 4e6 weeks until charac-

terization or storage. To get an overview of the genetic varia-

tion in the Nordic countries, 50 isolates from each country

were selected for genetic analysis. A hierarchical sampling

strategy was used at three macrospatial scales including

countries, districts and fields. The selection of isolates covered

all important potato growing regions in the Nordic countries,

as well as some areas with a more extensive production

(Fig 1). All selected isolates were from separate fields to avoid

analyzing clones within a field, but a few fields were located

close to each other. Mating type analysis of a larger number

of isolates from the fields covered in this study showed that

approximately 60 % of the isolates were A1 mating type in

each country. Furthermore, it was shown that both mating

types were present in 40 % of the fields where more than

one isolate was tested, indicating strong potential for sexual

reproduction (Lehtinen et al. 2008). Around a half (54 %) of

the isolates selected for genetic analysis were A1. Thus, the

mating type distribution among the tested isolates reflected

the mating type distribution in the total collection of isolates.

DNA extraction and SSR analysis

Mycelium for DNA extraction was grown on plates of pea agar

or amixture of pea and rye B agar (Lehtinen et al. 2008), for 2e4

weeks at 18 �C in the dark. Mycelium from plates was scraped

lysis of Phytophthora infestans populations in the Nordic Euro-011), doi:10.1016/j.funbio.2011.01.003

Genetic variability of Phytophthora infestans 3

off with a scalpel blade and blotted dry on filter paper. Themy-

celium was frozen in liquid nitrogen and disrupted using

a grinding mill (model MM301; Retsch, Haan, Germany) for

1.5 min at a vibration frequency of 30 oscillations per second.

DNAwas extracted using the DNeasy� Plant Mini Kit (Qiagen),

according to the manufacturer’s instructions.

Nine polymorphic SSR regions were amplified using PCR

with primers from previous studies: Pi02, Pi04, Pi16, Pi26,

Pi33 (Lees et al. 2006); 4B, 4G, G11, D13 (Knapova & Gisi 2002).

The primers were obtained from Applied Biosystems. For au-

tomated fragment analysis, one primer of each locus was la-

belled with a fluorescent dye (6-FAM, NED, PET, or VIC). Dyes

were assigned to loci in such a way that loci with the same

dye had nonoverlapping ranges of allele sizes. This allowed si-

multaneous loading of several loci amplification reactions

from one isolate, onto the capillary system (see below). Ampli-

fications were performed in 10-ml reaction volumes with 1.0

unit Taq DNA polymerase (Applied Biosystems), 200 mM

dNTP, 10 pmol each of forward and reverse primer, 1.5 mM

MgCl2, and 1 ml of 10� AmpliTaq buffer (500 mM KCl, 100 mM

TriseHCl pH 8.3 and 15 mM MgCl2). A total of 1 ml from the

DNA preparations was used as a template in each reaction.

The thermal cycling was carried out as follows: 95 �C for

4 min, followed by 35 cycles of 95 �C for 30 s, 59 �C for 30 s,

72 �C for 30 s, and a final extension of 7 min at 72 �C, beforecooling to 4 �C. All PCR reactions were performed with a Gen-

Amp PCR System 9700 (PE Applied Biosystems).

The fluorescently labelled PCR products were analyzed us-

ing an ABI3730 DNA Analyzer with 48 36 cm capillaries, using

Performance Optimized Polymer for 3730 DNA Analyzers

(POP-7; Applied Biosystems). 1 ml of 12.5 e 71-fold (depending

on the different markers) diluted PCR products were added

to a loading buffer containing 8.8 ml Hi-Di� formamide (Ap-

plied Biosystems), and 0.2 ml of GeneScan 500 LIZ size standard

(Applied Biosystems). Electrophoresis of the samples was car-

ried out at 66 �C and at 15 kV, for 20 min. The data was col-

lected using the software Data Collection v 2.0 (Applied

Biosystems), while GeneMapper v 4.0 (Applied Biosystems)

was used to derive the fragment length of the labelled DNA-

fragments using the known fragment lengths of the LIZ-

labelled marker peaks.

Allele size was determined by using the marker and by

comparison with ten reference isolates (C1e10) kindly sup-

plied by Drs Lees and Cooke (Lees et al. 2006). For some iso-

lates, three or four alleles were consistently identified at one

or more loci. This was mainly for locus Pi26, where as many

as 43 and 31 isolates gave three or four alleles respectively.

In addition, markers Pi02, Pi04, and G11 gave three alleles for

ten, three and one of the isolates respectively. This phenome-

non has also been reported by Lees et al. (2006), including for

five of the ten reference isolates that were used in this study.

Following the strategy described by Holmes et al. (2009), the in-

dividuals with more than two alleles at a locus were included

in the present analysis after systematically excluding the larg-

est allele(s). While exclusion of one or two alleles leads to an

underestimation of genetic variation at the loci in question,

it allowed us to consider more isolates in our analysis.

Multilocus genotypes were compiled for all isolates using

the seven loci Pi02, Pi04, Pi16, Pi26, Pi33, 4B, G11. If data were

missing in more than one of the seven loci for an isolate, it

Please cite this article in press as: Brurberg MB, et al., Genetic anapean countries reveals high genetic variability, Fungal Biology (2

was discarded from the genotype calculations. If an isolate

contained one missing locus it was considered to be identical

to another if the other six loci were identical. This could po-

tentially lead to an underestimation of genotypic diversity.

Data analysis

Deviations from HardyeWeinberg equilibrium at all loci were

tested with a chi-square test using POPGENE version 1.32 (Yeh

et al. 2000). In addition we calculated the index of association

(IA) to test for random recombination between pairs of all the

loci. This test quantifies the degree of recombination within

these loci through the formula: IA¼VO/VE� 1 (where VO is

the variation observed in the number of loci in which two in-

dividuals differ and VE is the expected variation). IA¼ 0

indicates frequent recombination events and IA values signif-

icantly larger than 0 indicates increasing association between

loci, and possibly also increasing clonality. Monte Carlo simu-

lations and parametric method were used to test the signifi-

cance of any difference between the calculated variance (VD)

and the variance expected at linkage equilibrium (VE). These

tests were carried out using the software LIAN (Haubold &

Hudson 2000).

Genotypic diversity was calculated by a normalized Shan-

non’s diversity index (Hs): Hs ¼ �PPilnPi=lnN, where Pi is

the frequency of the ithmultilocus genotype andN is the sam-

ple size. This diversity index corrects for differences in sample

size (Sheldon 1969). Values for Hs may range from 0 (single ge-

notype present) to 1 (each isolate in the sample has a different

genotype). The Shannon index could in principle lead to incor-

rect conclusions, particularly when diversity is low or sample

sizes differ as pointed out by Gr€unwald et al. (2003). However

in our study the diversity is high and the sample size is in prin-

ciple the same for all the four countries included.

Nei’s genetic distance (Nei 1978) was calculated using POP-

GENE (Yeh et al. 2000). Wemeasured the FST values to estimate

the genetic differentiation among all the four countries, also

using the POPGENE software (Yeh et al. 2000). The level of di-

vergence for each country was calculated as the mean value

of pairwise FST for each country against the remaining coun-

tries using the Arlequin software package, version 2000

(Schneider et al. 2000). The significance of FST values was

tested by 1023 permutations.

A Principal Coordinate (PCO) analysis was carried out to

perform ordination analysis and to classify and detect struc-

ture in the relationships between Phytophthora infestans iso-

lates using NTSYS-pc software, version 2.0 (Exeter Biological

Software, Setauket, NY).

Results

All the nine tested SSR loci were polymorphic for the selected

200 Nordic Phytophthora infestans isolates. Whereas seven of

the nine loci were detected in most of the isolates, the SSR

analysis failed to give results for loci D13 and 4G in 31 and

28 % of the isolates, respectively. The failure to produce re-

sults for these loci occurred with approximately the same fre-

quencies in isolates from all the four Nordic countries. These

lysis of Phytophthora infestans populations in the Nordic Euro-011), doi:10.1016/j.funbio.2011.01.003

4 M. B. Brurberg et al.

two loci were therefore omitted from the genotype

calculations.

The number of alleles detected at nine SSR loci in the 200

isolates analyzed was in total 49, ranging from two (at locus

Pi33) to nine (at locus D13 and G11) (Table 1). Isolates from

all four Nordic countries shared the most common alleles

across the analyzed SSR loci. For four of the nine loci (Pi02,

Table 1 e Allele frequencies for SSR markers in 192 P. infestan

SSR locus Allele Allel

Denmark(n¼ 49)

Finland(n¼ 50)

Pi02 152 0.22 0.06

158 0.01 0.00

160 0.26 0.08

162 0.51 0.86

164 0.00 0.00

166 0.00 0.00

174 0.00 0.00

176 0.00 0.00

Pi04 160 0.07 0.05

166 0.33 0.29

168 0.18 0.20

170 0.42 0.46

Pi16 158 0.00 0.00

160 0.00 0.00

174 0.17 0.42

176 0.83 0.58

Pi26 171 0.11 0.00

173 0.11 0.00

177 0.37 0.28

179 0.07 0.08

181 0.28 0.60

183 0.05 0.04

185 0.01 0.00

Pi33 203 0.97 0.77

206 0.03 0.23

4B 205 0.09 0.20

213 0.55 0.45

217 0.36 0.35

4G 161 0.04 0.07

163 0.93 0.77

165 0.04 0.16

D13 118 0.01 0.02

132 0.01 0.00

134 0.01 0.02

136 0.74 0.64

138 0.03 0.00

140 0.06 0.17

152 0.00 0.00

154 0.06 0.08

156 0.07 0.09

G11 142 0.07 0.18

148 0.09 0.00

152 0.00 0.00

154 0.14 0.08

156 0.11 0.37

158 0.00 0.00

160 0.32 0.11

162 0.26 0.26

164 0.00 0.00

a H ¼ 1�Px2j ; where xj is the frequency of the jth allele at the locus (Ne

Please cite this article in press as: Brurberg MB, et al., Genetic anapean countries reveals high genetic variability, Fungal Biology (2

Pi33, 4G, D13), one specific allele was detected in 70e80 % of

all the isolates, while for the other five loci (Pi04, Pi16, Pi26,

4B, G11) two specific alleles were detected in 62e95 % of all

the isolates. For locus Pi02, the most frequent allele (162)

was considerably less frequent in Denmark than the overall

population. While the most frequent alleles of loci Pi33 and

4G, were noticeably more frequent in Denmark than for all

s isolates from the Nordic countries collected in 2003

e frequency Genediversity (H)a

Norway(n¼ 50)

Sweden(n¼ 50)

Overall(n¼ 199)

0.01 0.08 0.09 0.48

0.01 0.01 0.01

0.14 0.10 0.14

0.65 0.79 0.70

0.02 0.00 0.01

0.01 0.02 0.01

0.07 0.00 0.02

0.09 0.00 0.02

0.09 0.07 0.07 0.68

0.35 0.30 0.32

0.09 0.26 0.18

0.47 0.37 0.43

0.07 0.00 0.02 0.47

0.14 0.00 0.03

0.31 0.22 0.28

0.49 0.78 0.67

0.03 0.01 0.04 0.69

0.03 0.01 0.04

0.25 0.35 0.31

0.13 0.14 0.11

0.50 0.39 0.45

0.06 0.09 0.06

0.00 0.01 0.01

0.70 0.78 0.80 0.31

0.30 0.22 0.20

0.24 0.22 0.19 0.63

0.29 0.50 0.45

0.47 0.28 0.36

0.09 0.07 0.07 0.32

0.77 0.78 0.80

0.14 0.16 0.13

0.03 0.04 0.03 0.47

0.00 0.03 0.01

0.00 0.08 0.03

0.86 0.65 0.72

0.00 0.04 0.02

0.02 0.03 0.06

0.02 0.05 0.02

0.06 0.09 0.07

0.02 0.01 0.05

0.09 0.11 0.11 0.78

0.04 0.05 0.05

0.02 0.01 0.01

0.03 0.02 0.07

0.42 0.25 0.30

0.01 0.01 0.01

0.04 0.11 0.14

0.34 0.40 0.32

0.00 0.04 0.01

i 1978).

lysis of Phytophthora infestans populations in the Nordic Euro-011), doi:10.1016/j.funbio.2011.01.003

Genetic variability of Phytophthora infestans 5

the Nordic countries together. Of the rarer alleles, five were

detected only in Norway, at loci Pi02 and Pi16, where they

were detected with relative low frequencies (0.01e0.14). The

five ‘Norwegian alleles’ were observed in 14 isolates, originat-

ing from different regions of Norway, meaning that each of

these isolates possessed at least two of such alleles. One low

frequency private allele at locus G11 was detected in Sweden.

The allele frequencies were processed further to consider

the combination of different alleles at each separate SSR lo-

cus, and gene diversities for each locus were calculated. The

gene diversity for single loci for all countries together (overall)

ranged from 0.31 to 0.78 (Table 1), and the average for all loci

was 0.54 which is 71 % of the maximum gene diversity based

on the number of observed alleles in all Nordic countries (re-

sults not shown). There was some variation in gene diversity

for each locus in each of the countries, but the average gene

diversity for each country was approximately the same

(0.48e0.54) (results not shown).

The 191 isolates in which we were able to score six loci or

more, produced in total 169 distinct multilocus genotypes,

meaning that most genotypes were detected only once. Five

genotypes occurred twice, one genotype was detected four

times (three in Sweden and one in Denmark) and two geno-

types occurred eight times. Of the two most frequent geno-

types, one occurred only in Denmark (seven of the eight

occurrences in one region) and the other was detected twice

in Finland (in the same region) and six times in four different

regions in Norway. The genotypic diversities, quantified by

a normalized Shannon’s diversity index (Hs), were larger

than 0.9 for all the four individual countries and for the Nordic

countries together 0.95 (Table 2).

The chi-square test (result not shown) failed to reject the

null hypothesis indicating that all loci were under

HardyeWeinberg equilibrium. Furthermore, the frequency

of recombination was estimated by calculating the

Table 2 e Genotypic diversity (single and multilocus) of P. infes

SSR locus Number ofgenotypes

N

Denmark(n¼ 49)

Pi02 12 198 0.416

Pi04 6 191 0.262

Pi16 6 186 0.182

Pi26 15 195 0.445

Pi33 3 194 0.062

4B 6 199 0.362

4G 5 144 0.123

D13 16 139 0.341

G11 25 168 0.647

Multilocusd 169 0.903

3.437b

0.945c

a Normalized Shannon index for all single loci and mulitilocus genotype

each country name.

b Non-normalized Shannon index for multilocus genotypes in each coun

c Shannon index divided by number of genotypes.

d The multilocus genotype is based on the seven markers Pi02, Pi04, Pi1

Finland, Norway, and Sweden respectively.

Please cite this article in press as: Brurberg MB, et al., Genetic anapean countries reveals high genetic variability, Fungal Biology (2

standardized index of association (IAS ) between loci. The IA

S

value for the 191 isolates analyzed was 0.0478, which is

not significantly different from zero (P< 0.001) indicating

that the isolates were in linkage equilibrium and hence

showing evidence of a high rate of sexual reproduction

among the isolates.

Genetic differentiationwithin the Nordic countries asmea-

sured by FST was 0.04. Pairwise measures of genetic distance

ranged from 0.025 to 0.126 (Table 3). Pairwise FST was calcu-

lated for each pair of countries over seven loci. The results

showed very low differentiation between all pairs of countries

(Table 3). The PCO analysis indicated no association between

the SSR fingerprint (or genetic diversity) and geographical or-

igin (Fig 2).

Discussion

Using nine SSR markers, including several newly developed

ones (Lees et al. 2006), we detected in total 49 alleles in the Phy-

tophthora infestans populations from Denmark, Finland, Nor-

way, and Sweden. Most of these alleles have previously been

detected in other P. infestans populations (www.eucablight.

org). The frequencydistributionof the alleles varied somewhat

between the Nordic countries and was also slightly different

from what has been observed previously in other countries.

The lowFST valueof 0.04 indicates that themajorityof variation

is found within, and not among, the four Nordic countries. In-

dividualswithin countries are likely to be genetically different,

but each country contains the same complement of alleles in

similar frequencies. The overall picture emerging from the re-

sults of the allele frequencies is that the isolates sampled in the

four Nordic countries come from a common population of

P. infestans. However, there is in general no exchange of seed

potatoes between the Nordic countries.

tans isolates from the Nordic countries collected in 2003

Genotypic diversitya

Finland(n¼ 50)

Norway(n¼ 50)

Sweden(n¼ 50)

Overall(n¼ 199)

0.207 0.355 0.291 0.275

0.311 0.211 0.299 0.211

0.261 0.331 0.223 0.212

0.379 0.445 0.507 0.375

0.199 0.214 0.175 0.138

0.425 0.460 0.373 0.301

0.266 0.303 0.206 0.184

0.403 0.224 0.479 0.321

0.591 0.499 0.624 0.491

1.000 0.936 0.966 0.954

3.912b 3.644b 3.721b 5.013b

1.000c 0.969c 0.989c 0.977c

s. The number of isolates analyzed from each country is given below

try.

6, Pi26, Pi33, 4B, G11 from 45, 50, 49 and 47 isolates from Denmark,

lysis of Phytophthora infestans populations in the Nordic Euro-011), doi:10.1016/j.funbio.2011.01.003

Table 3 e Matrix of genetic distance and populationdifferentiation for Phytophthora infestans populationsfrom the Nordic countries, based on analysis of sevenpolymorphic loci. Values below the diagonal are Nei’sgenetic distance; values above the diagonal are estimatedFST values for each pairwise comparison of isolates,sampled from Norway, Denmark, Finland, and Sweden

Regions Norway Sweden Finland Denmark

Norway 0.03819a 0.03189a 0.04091a

Sweden 0.051 0.04819a 0.04364a

Finland 0.025 0.026 0.04574a

Denmark 0.126 0.053 0.101

a Significant at P� 0.001.

6 M. B. Brurberg et al.

The PCO analysis showed that there was no distinguish-

able pattern of clustering of isolates from a certain country.

The lack of geographic clustering was also supported by the

low FST values found among the countries (Table 3). The ab-

sence of a geographic structure suggests a relatively recent ex-

pansion of a single diverse population.

The SSR analysis of P. infestans isolates from the Nordic

countries, Denmark, Finland, Norway, and Sweden, revealed

a high level of variation with respect to genotypes. Most iso-

lates had a distinct genetic fingerprint with 169 genotypes

detected among the 191 isolates in which we were able to

score six SSR loci or more. No particular genotypes dominated

in any of the four countries, but one genotype occurred seven

times in the same district in Denmark. Our data shows that

genotypic variation in P. infestans anno 2003 is even larger

than the variation found in most previous studies. A study

of isolates collected in Finland and Norway in the period

1992e1996 using RFLP (RG57) fingerprinting yielded genotypic

diversities (Hs) for the Norwegian and Finnish isolates of 0.75

and 0.83, respectively (Brurberg et al. 1999). A study of early

5,0-

4,0-

3,0-

2,0-

1,0-

0

1,0

2,0

3,0

4,0

001,0-2,0-3,0-4,0-

sixaOCP

PC

O a

xis 2

(2

9.9

6%

)

yawroNnedewSdnalniFkramneD

Fig 2 e PCO analysis of 200 Phytophthora infestans isolates from

versity of seven polymorphic loci with totally 37 alleles.

Please cite this article in press as: Brurberg MB, et al., Genetic anapean countries reveals high genetic variability, Fungal Biology (2

infection in a single field in southwest Sweden using six SSR

loci including 4B, G11, and Pi16 used in the current study

revealed unique genotypes for five out of the six discrete dis-

ease foci studied, and the genotypic diversity (Hs) was 0.54

(Widmark et al. 2007). A comparison of the genetic variation

of P. infestans isolates from organic potato crops in Norway,

France, Switzerland and the United Kingdom that was con-

ducted in 2001 using AFLP showed that genetic variation in

Norway was higher than in the other countries (Flier et al.

2007). While it should be noted that observed genetic diversi-

ties to some extent depend on markers used and the geo-

graphic scales of the analyses, the overall picture provided

by the data referred to above indicates a relatively high genetic

diversity in the Nordic countries.

High levels of genotypic diversity such as those found in

the present study have previously been described for P. infes-

tans populations in other North European countries

(Sujkowski et al. 1994; Drenth et al. 1994) as well as in the

two regions that are currently considered as possible origins

of P. infestans, central Mexico (Goodwin et al. 1992) and the

SouthAmericanAndes (Gom�ez-Alpizar et al. 2007). In contrast,

P. infestans populations in other parts of the world, including

north-western Mexico (Goodwin et al. 1992), Ecuador (Forbes

et al. 1997), Asia (Koh et al. 1994; Le et al. 2008), North America

(Goodwin et al. 1995) and other parts of Europe (Lebreton &

Andrivon 1998; Day et al. 2004; Cooke et al. 2006; Flier et al.

2007; Montarry et al. 2008, 2010) are dominated by clonal line-

ages. In some of these latter countries, most local populations

consist of a single clonal lineage (either A1 or A2), which re-

stricts sexual reproduction.

So far, there are few published studies that employ the

same large set of markers used here. In a recent study aimed

at testing the utility of SSR markers for analysis of P. infestans

populations, 77 isolates from the UK and 13 from the rest of

the world were analyzed for genetic variation using 12 SSR

6,05,04,03,02,01,

)%98.93(1

Norway, Sweden, Finland, and Denmark based on SSR di-

lysis of Phytophthora infestans populations in the Nordic Euro-011), doi:10.1016/j.funbio.2011.01.003

Genetic variability of Phytophthora infestans 7

markers (Lees et al. 2006), including all markers used in the

present study, except 4G. Even though these isolates were se-

lected in anticipation of a high level of diversity and were

scoredwith up to 12markers, only 68 genotypeswere detected

among the 90 isolates (Lees et al. 2006). Increasing the number

of markers normally increases the number of genotypes, and

in the study by Lees et al. (2006), maximum resolution was

achieved by using ten loci. Increasing the number of markers

from seven to ten increased the number of genotypes by ap-

proximately 30 % in their selected populations (Lees et al.

2006). This shows that the Nordic P. infestans populations

have an extremely high genotypic diversity compared to other

populations studied,when using the same tools for analysis. It

is possible that our findings to a certain degree reflect our

sampling strategy (one isolate from each field), but it still

seems apparent from our study that the Nordic P. infestans

populations are genetically highly variable.

What is less clear is the cause of the high level of genetic

variation compared to many other countries. Judging from

the recent mating type distributions, which showed that ap-

proximately 60 % of the isolates were of mating type A1 in

each country and that both mating types were often found

in the same fields (Lehtinen et al. 2008) there is clearly a poten-

tial for frequent sexual reproduction. Previous studies indi-

cate that both mating types have been present in the Nordic

countries since around 1992. The mating type frequencies

have been monitored most carefully in Finland, in which A2

increased gradually from approximately 20 % in 1992 to 50 %

in 2000, so the potential for sexual recombination is not en-

tirely new. It is well known that sexual recombination in-

creases genotype diversity in populations, since it creates

novel recombinants. A high level of recombination is sug-

gested by the low index of association and the fact that no

loci are in linkage disequilibrium. The hypothesis that sexual

reproduction greatly contributes to the variation in the popu-

lation is also supported by the fact that the number of alleles

at each locus and their frequencies are not very different from

what we see in for example UK where the population is

regarded as mainly clonal (Lees et al. 2006).

In the first report concerning the high level of genetic var-

iability in Norway and Finland compared to some other Euro-

pean countries it was speculated that the increased rate of

sexual reproduction could be caused by climatic differences

between the Nordic countries and other European countries

(Brurberg et al. 1999). Comparatively cool summers slow the

infection process and the deterioration of tissue (Erwin &

Ribeiro 1996) and such conditions may promote mating and

oospore formation. Romero-Montes et al. (2008) concluded

from an experiment in Mexico that oospore formation is

more dependent on environmental factors (rain) and on the

induction of slow epidemics (disease management), than on

the geneticmakeup of the host. In a climatewith cold winters,

inoculum from volunteers and dump piles are less important

since survival of P. infestans on tubers is largely dependent on

the viability of the tuber tissue as well as the pathogen’s phys-

iological capability of surviving low temperatures or freezing

conditions. As a result oospores may be proportionally more

important as a source of inoculum resulting in a population

more influenced by sexual recombination. Frost in the soil

might also synchronize the germination of oospores with

Please cite this article in press as: Brurberg MB, et al., Genetic anapean countries reveals high genetic variability, Fungal Biology (2

the planting of the potato crop (Widmark et al. 2007) and in

this way further increases the importance of oospores as inoc-

ulum of P. infestans. Oospores have indeed been detected in

numerous studies in the Nordic countries and are considered

important inoculum sources (Andersson et al. 2009).

Othermechanisms, such asmutation andmitotic recombi-

nation, may also contribute to genetic variation in the P. infes-

tans populations. Asexual progenies of P. infestans have

previously been shown to differ from their parents in several

characteristics such as aggressiveness, growth rate, colony

morphology and virulence (Caten & Jinks 1968; Abu-El

Samen et al. 2003). Mitotic gene conversion was observed to

occur at remarkably high frequencies in Phytophthora sojae

(Chamnanpunt et al. 2001) documenting potential for rapid

generation of variation. In general populations with large ef-

fective sizes, such as populations of P. infestans, tend to have

higher gene diversity, asmore alleles can emerge throughmu-

tation and fewer alleleswill be lost due to randomgenetic drift

(Hartl & Clark 1997). However, there is no known reason that

these mechanisms should contribute to more diversity in

P. infestans in the Nordic region than elsewhere.

This study, conducted with isolates of P. infestans taken

from different fields early in the late blight epidemic indicates

that the pathogen population is extremely diverse, with 169

unique multilocus genotypes detected among 191 isolates.

While allele frequencies are similar in the different Nordic

countries, only a few genotypes occurred more than once.

This study, together with others on the equal numbers of

both mating types, presence of oospores in field material, in-

oculum sources in the soil, and variability between andwithin

infection foci, indicate that sexual reproduction is a major

determinant of the population structure of P. infestans in the

Nordic countries.

Acknowledgements

We thank Alison Lees and David Cooke (SCRI, Dundee, Scot-

land) for information prepublication on SSR markers, DNA

from control isolates as well as discussions on scoring of

SSR markers. We are also grateful to Grete Lund for technical

assistance. This work was part of the NorPhyt project, sup-

ported by the Nordic Joint Committee for Agricultural

Research.

r e f e r e n c e s

Abu-El Samen FM, Secor GA, Gudmestad NC, 2003. Genetic vari-ation among asexual progeny of Phytophthora infestans de-tected with RAPD and AFLP markers. Plant Pathology 52:314e325.

Andersson B, Sandstr€om M, Str€omberg A, 1998. Indications of soilborne inoculum of Phytophthora infestans. Potato Research 41:305e310.

Andersson B, Widmark A-K, Yuen JE, Nielsen B, Ravnskov S,Kessel GJT, Evenhuis A, Turkensteen LJ, Hansen JG,Lehtinen A, Hermansen A, Brurberg MB, Nordskog B, 2009. Therole of oospores in the epidemiology of potato late blight. ActaHorticulturae 834: 61e68.

lysis of Phytophthora infestans populations in the Nordic Euro-011), doi:10.1016/j.funbio.2011.01.003

8 M. B. Brurberg et al.

Aylor DE, 2003. Spread of plant disease on a continental scale: roleof aerial dispersal of pathogens. Ecology 84: 1989e1997.

Brurberg MB, Hannukkala A, Hermansen A, 1999. Genetic vari-ability of Phytophthora infestans in Norway and Finland as re-vealed by mating type and fingerprint probe RG57. MycologicalResearch 103: 1609e1615.

Caten CE, Jinks JL, 1968. Spontaneous variability of single isolatesof Phytophthora infestans. I. Cultural variation. Canadian Journalof Botany 46: 329e348.

Chamnanpunt J, Shan WX, Tyler BM, 2001. High frequency mi-totic gene conversion in genetic hybrids of the oomycetePhytophthora sojae. Proceedings of the National Academy of Sciencesof the United States of America 98: 14530e14535.

Cooke DEL, Lees AK, 2004. Markers, old and new, for examiningPhytophthora infestans diversity. Plant Pathology 53: 692e704.

Cooke LR, Carlisle DJ, Donaghy C, Quinn M, Perez FM, Deahl KL,2006. The Northern Ireland Phytophthora infestans population1998e2002 characterized by genotypic and phenotypicmarkers. Plant Pathology 55: 320e330.

Day JP, Wattier RAM, Shaw DS, Shattock RC, 2004. Phenotypic andgenotypic diversity in Phytophthora infestans on potato in GreatBritain, 1995e98. Plant Pathology 53: 303e315.

Drenth A, Tas ICQ, Govers F, 1994. DNA fingerprinting uncoversa new sexually reproducing population of Phytophthora infes-tans in the Netherlands. European Journal of Plant Pathology 100:97e107.

Drenth A, Janssen EM, Govers F, 1995. Formation and survival ofoospores of Phytophthora infestans under natural conditions.Plant Pathology 44: 86e94.

Erwin DC, Ribeiro OK, 1996. Phytophthora Diseases Worldwide. APSPress, St. Paul, MN, USA.

Flier WG, Kroon LPNM, Hermansen A, van Raaij HMG, Speiser B,Tamm L, Fuchs JG, Lambion J, Razzaghian J, Andrivon D,Wilcockson S, Leifert C, 2007. Genetic structure and pathoge-nicity of populations of Phytophthora infestans from organicpotato crops in France, Norway, Switzerland and the UnitedKingdom. Plant Pathology 56: 562e572.

Forbes GA, Escobar XC, Ayala CC, Revelo J, Ordo~nez ME, Fry BA,Doucett K, Fry WE, 1997. Population genetic structure of Phy-tophthora infestans in Ecuador. Phytopathology 87: 375e380.

Fry W, 2008. Phytophthora infestans: the plant (and R gene) de-stroyer. Molecular Plant Pathology 9: 385e402.

Gom�ez-Alpizar L, Carbone I, Ristaino JB, 2007. An Andean originof Phytophthora infestans inferred from mitochondrial and nu-clear gene genealogies. Proceedings of the National Academy ofSciences of the United States of America 104: 3306e3311.

Goodwin SB, Spielman LJ, Matuszak JM, Bergeron SN, Fry WE,1992. Clonal diversity and genetic differentiation of Phytoph-thora infestans populations in northern and central Mexico.Phytopathology 82: 955e961.

Goodwin SB, Cohen BA, Fry WE, 1994. Panglobal distribution ofa single clonal lineage of the Irish potato famine fungus. Pro-ceedings of the National Academy of Sciences of the United States ofAmerica 91: 11591e11595.

Goodwin SB, Sujkowski LS, Dyer AT, Fry BA, Fry WE, 1995. Directdetection of gene flow and probable sexual reproduction ofPhytophthora infestans in Northern North America. Phytopa-thology 85: 473e479.

Gr€unwald NJ, Goodwin SB, Milgroom MG, Fry WE, 2003. Analysisof genotypic diversity data for populations of microorganisms.Phytopathology 93: 738e746.

Hartl DL, Clark AG, 1997. Principles of Population Genetics, 3rd edn.Sinauer Associates, Sunderland, MA.

Haubold B, Hudson RR, 2000. LIAN 3.0: detecting linkage dis-equilibrium in multilocus data. Bioinformatics 16: 847e848.

Hermansen A, Hannukkala A, Nærstad RH, Brurberg MB, 2000.Variation in populations of Phytophthora infestans in Finland

Please cite this article in press as: Brurberg MB, et al., Genetic anapean countries reveals high genetic variability, Fungal Biology (2

and Norway: mating type, metalaxyl resistance and virulencephenotype. Plant Pathology 49: 11e22.

Hohl HR, Iselin K, 1984. Strains of Phytophthora infestans fromSwitzerland with A2 mating type behaviour. Transactions of theBritish Mycological Society 83: 529e531.

Holmes GD, James EA, Hoffmann AA, 2009. Divergent levels ofgenetic variation and ploidy among populations of the rareshrub, Grevillea repens (Proteaceae). Conservation Genetics 10:827e837.

Knapova G, Gisi U, 2002. Phenotypic and genotypic structure ofPhytophthora infestans populations on potato and tomato inFrance and Switzerland. Plant Pathology 51: 641e653.

Koh YJ, Goodwin SB, Dyer AT, Cohen BA, Ogoshi A, Sato N,Fry WE, 1994. Migrations and displacements of Phytophthorainfestans in East Asian countries. Phytopathology 84: 922e927.

Le VH, Ngo XT, Brurberg MB, Hermansen A, 2008. Characterisa-tion of Phytophthora infestans populations from Vietnam. Aus-tralasian Plant Pathology 37: 592e599.

Lebreton L, Andrivon D, 1998. French isolates of Phytophthora in-festans from potato and tomato differ in phenotype and ge-notype. European Journal of Plant Pathology 104: 583e594.

Lees AK, Wattier R, Shaw DS, Sullivan L, Williams NA,Cooke DEL, 2006. Novel microsatellite markers for the anal-ysis of Phytophthora infestans populations. Plant Pathology 55:311e319.

Lehtinen A, Hannukkala A, Andersson B, Hermansen A, Le VH,Nærstad R, Brurberg MB, Nielsen BJ, Hansen JG, Yuen J, 2008.Phenotypic variation in Nordic populations of Phytophthorainfestans in 2003. Plant Pathology 57: 227e234.

Montarry J, Glais I, Corbiere R, Andrivon D, 2008. Adaptation tothe most abundant host genotype in an agriculturalplantepathogen system e potato late blight. Journal of Evolu-tionary Biology 21: 1397e1407.

Montarry J, Andrivon D, Glais I, Corbiere R, Mialdea G, Delmotte F,2010. Microsatellite markers reveal two admixed geneticgroups and an ongoing displacement within the French pop-ulation of the invasive plant pathogen Phytophthora infestans.Molecular Ecology 19: 1965e1977.

Nei M, 1978. Estimation of average heterozygosity and geneticdistance from a small number of individuals. Genetics 89:583e590.

Romero-Montes G, Lozoya-Salda~na H, Mora-Aguilera G, Fern�an-dez-Pavia S, Gr€unwald NJ, 2008. Environmental and slow epi-demics favor oosporulation of Phytophthora infestans Mont. DeBary, on potato leaves in the Toluca Valley, M�exico. AmericanJournal of Potato Research 85: 101e109.

Schneider R, Roessli D, Excoffier L, 2000. Arlequin: a Software forPopulation Genetics Data Analysis, Version 2.000. Genetic andBiometry Laboratory, Department of Anthropology, Universityof Geneva, Switzerland.

Sheldon AL, 1969. Equitability indices: dependence on the speciescount. Ecology 50: 466e467.

Sujkowski LS, Goodwin SB, Dyer AT, Fry WE, 1994. Increased ge-notypic diversity via migration and possible occurrence ofsexual reproduction of Phytophthora infestans in Poland. Phyto-pathology 84: 201e207.

Turkensteen LJ, Flier WG, Wanningen R, Mulder A, 2000. Produc-tion, survival and infectivity of oospores of Phytophthora in-festans. Plant Pathology 49: 688e696.

Widmark A-K, Andersson B, Cassel-Lundhagen A, Sandstr€om M,Yuen JE, 2007. Phytophthora infestans in a single field in south-west Sweden early in spring: symptoms, spatial distributionand genotypic variation. Plant Pathology 56: 573e579.

Yeh FC, Yang R, Boyle TJ, Ye Z, Xiyan JM, 2000. PopGene32. Micro-soft Windows-based Freeware for Population Genetic Analysis,Version 1.32. Molecular Biology and Biotechnology Centre,University of Alberta, Edmonton.

lysis of Phytophthora infestans populations in the Nordic Euro-011), doi:10.1016/j.funbio.2011.01.003